Sperm cells are exposed to many different environments in both male and female reproductive tracts. To function in disparate milieus, sperm have evolved into highly polarized, multifunctional cells whose surface is comprised of several large domains. Much of this membrane polarization occurs as sperm pass through the epididymis. For example, while in the epididymis, the plasma membrane surrounding the sperm head polarizes several proteins into either the posterior (PHD) or anterior (AHD) head domain. In many somatic cell systems, membrane polarization precedes a change in the cellular function. Thus redistribution of sperm proteins may be an important prerequisite for functional development of sperm seen in the epididymis. Likewise, maintaining a polarized membrane is critical if these functions are to continue. We are interested in understanding the molecular mechanisms that enable sperm to maintain a polarized membrane. To study this, we have focused on the transmembrane protein fertilin. Fertilin is found distributed over the whole head of testicular sperm. But on sperm that have moved through the epididymis (cauda sperm), it is only found in the PHD. We examined how fertilin is retained within the whole head domain of testicular sperm, and on just the PHD of cauda sperm. Using fluorescence redistribution after photobleaching (FRAP) analysis, we found fertilin is highly restricted from moving within the lipid bilayer of both testicular and cauda sperm, and thus does not diffuse out of these domains. However, when we examined capacitated cauda sperm or acrosome-reacted cauda sperm, fertilin was highly mobile, yet still retained within the PHD. In this proposal we have designed experiments to try answering the following: 1) What is the mechanism(s) responsible for restricting fertilin's diffusion in PHD of cauda sperm? 2) What is the signal during capacitation that causes fertilin to become mobile within the membrane? 3) Is fertilin physically modified during capacitation and/or the acrosome reaction, and if so how? We anticipate these studies will advance understanding of the sperm's cellular architecture and its relationship to cellular function. Information gained may also lead to development of novel male contraceptives and/or treatments for infertility.